Kinetics of multi-step reactions: insights into the mechanisms of P450 enzymes

Title:  Kinetics of multi-step reactions: insights into the mechanisms of P450 enzymes

Speaker:  Prof F. Peter Guengerich, Vanderbilt University, School of Medicine, Nashville, Tennessee, U.S.A

Abstract:  Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in drug metabolism, as well as many oxidations of vitamins, steroids, natural products, and industrial chemicals. A number of P450s catalyze muti-step oxidation reactions, and one issue has been the degree to which these are processive or distributive processes, i.e. whether or not the intermediate products dissociate from the enzyme before subsequent steps occur. This question has been addressed with several of the human P450s that oxidize steroids. P450 19A1 is the steroid aromatase that forms estrogens, and it was shown to be a distributive enzyme. P450 17A1 catalyzes the 17a-hydroylation of progesterone and pregnenolone, followed by a “lyase” reaction to form androgens. This processive was shown to involve a partially processive pathway, where a fraction of the 17a-hydroxy steroid product dissociates from the enzyme. P450 Family 51 enzymes catalyze the 14a-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51A1 catalyzes the 3-step, 6-electron oxidation of lanosterol or 24,25-dihydrolanosterol to form (4b,5a)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol, FF-MAS) via 14a-alcohol and 14a-aldehyde intermediates. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, with koff rates of P450 51A1-dihydrolanosterol and the 14a-alcohol and 14a-aldehyde complexes being 1-2 orders of magnitude less than the forward rates of competing oxidations. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Bio: Prof F. Peter Guengerich received his B.S. from the University of Illinois in 1970 and a Ph.D. (Biochemistry) from Vanderbilt University in 1993 (with Prof. H. P. Broquist).  Following two years of postdoctoral training at the University of Michigan (with Prof. M. J. Coon), he joined the faculty at Vanderbilt as Assistant Professor of Biochemistry.  He became (full) Professor in 1983 and was Director of the Vanderbilt Center in Molecular Toxicology from 1980-2011.  Prof. Guengerich is an enzymologist and his interests have been in the characterization of cytochrome P450 enzymes and the metabolism and bioactivation of drugs and toxic chemicals.  He has published 759 refereed papers, 317 invited reviews, and 138 published proceedings and was the 46th most highly cited biomedical scientist for articles published in the 1990s.  He is one of the most highly cited authors in the fields of biochemistry (and toxicology) today. Prof. Guengerich is a Fellow of the American Chemical Society, the American Society for Biochemistry and Molecular Biology, and the American Society for Pharmacology and Experimental Therapeutics and has received major awards from these and other scientific societies. He has directed the training of 22 graduate students and 141 postdoctoral fellows. In 2000 he received the first Vanderbilt School of Medicine Medal given for Mentoring Postdoctoral Fellows or Residents in the Research Setting, and subsequently this biennnial award was named for him. Prof. Guengerich served as an Associate Editor, Deputy Editor, and Interim Editor-in-Chief of The Journal of Biological Chemistry from 2006-2022.